Method of identifying changes in biopolymers

Abstract

The current invention relates to a method of identifying changes in biopolymers, especially in chromosomal DNA, using two or more different sets of labelled detector molecules, as well as to a diagnostic kit for detecting these changes.

1. A method of identifying differences between biopolymers, the method comprising the steps:
 a) providing at least one pair of different sets of labeled detector molecules wherein the different sets of labeled detector molecules of one pair are specifically bondable to a certain region in said biopolymers; and the labels of the one set of labeled detector molecules of said one pair differs from the labels of another one set of labeled detector molecules of said one pair; 
 b) exposing said labeled detector molecules to said biopolymers under conditions permitting bonding reactions to occur between said labeled detector molecules and said biopolymers; wherein said one set of labeled detector molecules of said one pair binds to a certain region of the biopolymer overlapping with the certain region bound by said another one set of labeled detector molecules of said one pair; 
 c) recording the presence, intensity and intensity ratios of the labeled detector molecules at selected regions of said biopolymers by scanning the biopolymers in longitudinal direction using a scanning device; 
 d) evaluating the recorded intensities and intensity ratios, whereby the biopolymer is divided into a number of sections and by means of an appropriate calculation program, a false color is assigned to each of these sections based on the relative intensity ratios; and 
 e) identifying differences between said biopolymers by comparing the result to that obtained for a different biopolymer, wherein the bonded labeled detector molecules of one set display a continuously changing label-signal intensity along the longitudinal direction of the biopolymer. 
 
     
     
       2. The method of  claim 1 , wherein the bonded labeled detector molecules of each of the different sets of one pair display a continuously changing label-signal intensity along the longitudinal direction of the biopolymer. 
     
     
       3. The method of  claim 2 , wherein the displayed continuously changing label-signal intensity along the longitudinal direction results from a continuously changing concentration of each of the different sets of bonded labeled detector molecules of one pair along the longitudinal direction of the biopolymer. 
     
     
       4. The method of  claim 3 , wherein the continuously changing concentration of each of the different sets of bonded labeled detector molecules of one pair is distributed along the longitudinal direction in a Gaussian distribution. 
     
     
       5. The method of  claim 1 , wherein the biopolymers are immobilized at least before step (b). 
     
     
       6. The method of  claim 5 , wherein the biopolymers are immobilized on a carrier or in a matrix. 
     
     
       7. The method of  claim 1 , wherein said bonding reactions between each of said labeled detector molecules and said biopolymer are carried out simultaneously or successively. 
     
     
       8. The method of  claim 1 , wherein said bonding reaction in step (b) is selected from the group consisting of a nucleic acid hybridization and an antigen/antibody reaction. 
     
     
       9. The method of  claim 8 , wherein said nucleic acid hybridization is an in situ hybridization. 
     
     
       10. The method of  claim 1 , wherein said biopolymers are selected from the group consisting of nucleic acids and polypeptides. 
     
     
       11. The method of  claim 10 , wherein said nucleic acids are DNA or RNA. 
     
     
       12. The method of  claim 10 , wherein said nucleic acids are chromosomal DNA. 
     
     
       13. The method of  claim 1 , wherein the labeled detector molecules are selected from the group consisting of nucleic acids and antibodies. 
     
     
       14. The method of  claim 13 , wherein said different nucleic acids are selected from different chromosome region-specific DNA libraries. 
     
     
       15. The method of  claim 1 , wherein the label comprises a fluorescent dye. 
     
     
       16. The method of  claim 1 , wherein said step (a) further comprises providing at least one set of a localized calibrating probe, said probe comprising calibrating labels. 
     
     
       17. The method of  claim 16 , wherein said calibrating labels comprise all of said labels of said labeled detector molecules of the different sets of at least one pair. 
     
     
       18. The method of  claim 1 , wherein said step (a) further comprises providing a number of localized calibrating probes, said number being one less than the total number of said labels in said labeled detector molecules, each of said probes comprising two labels; and said step (d) further comprises correcting registration errors between individual images corresponding to individual labels, said registration errors being introduced by changing filters between the acquisition of said individual images; said correcting step being achieved by pairwise comparison of the positions of the two labels of said calibrating probes. 
     
     
       19. The method of  claim 18 , wherein said step (d) further comprises forming images of said biopolymers; and aligning said images with respect to said bondings, thereby providing positional correction for said bondings. 
     
     
       20. The method of  claim 19 , wherein said calibrating probes are used for positional correction of said bondings. 
     
     
       21. The method of  claim 18 , wherein said step (a) further comprises providing a plurality of said calibrating probes; and said step (d) further comprises correcting positional transformations of said bondings by comparison of the position of the labels of said calibrating probes. 
     
     
       22. The method of  claim 21 , wherein said step of correcting is automatic. 
     
     
       23. The method of  claim 21 , wherein said labels of said calibrating probes have known or reproducible constant intensity whereby the signal intensities of all of said labels can be standardized. 
     
     
       24. The method of  claim 23 , wherein said calibrating probes are fluorescence-labeled probes. 
     
     
       25. The method of  claim 23 , wherein said calibrating probes are fluorescence-labeled particles. 
     
     
       26. A method of identifying differences between biopolymers, the method comprising the steps:
 a) providing at least two different sets of labeled detector molecules wherein at least two sets of said labeled detector molecules at a time are specifically bondable to a certain region in said biopolymers; and the labels of said labeled detector molecules of one of said at least two sets differ from the labels of said labeled detector molecules of another of said at least two sets; 
 b) exposing said labeled detector molecules to said biopolymers under conditions permitting bonding reactions to occur between said labeled detector molecules and said biopolymers; wherein the labeled detector molecules of said one of said at least two sets binds to a certain region of the biopolymer overlapping with the certain region bound by said labeled detector molecules of another of said at least two sets; 
 c) recording the presence, intensity and intensity ratios of the labeled detector molecules at selected regions of said biopolymers by scanning the biopolymers in longitudinal direction using a scanning device; 
 d) evaluating the recorded intensities and intensity ratios, whereby the biopolymer is divided into a number of sections and by means of an appropriate calculation program, a false color is assigned to each of these sections based on the relative intensity ratios; and 
 e) identifying differences between said biopolymers by comparing the result to that obtained for a different biopolymer, wherein the bonded labeled detector molecules of one set display a continuously changing label-signal intensity along the longitudinal direction of the biopolymer. 
 
     
     
       27. The method of  claim 26 , wherein the bonded labeled detector molecules of each of the different sets display a continuously changing label-signal intensity along the longitudinal direction of the biopolymer. 
     
     
       28. The method of  claim 27 , wherein the displayed continuously changing label-signal intensity along the longitudinal direction results from a continuously changing concentration of each of the different sets of bonded labeled detector molecules along the longitudinal direction of the biopolymer. 
     
     
       29. The method of  claim 28 , wherein the continuously changing concentration of each of the different sets of bonded labeled detector molecules is distributed along the longitudinal direction in a Gaussian distribution. 
     
     
       30. The method of  claim 26 , wherein the biopolymers are immobilized at least before step (b). 
     
     
       31. The method of  claim 30 , wherein the biopolymers are immobilized on a carrier or in a matrix. 
     
     
       32. The method of  claim 26 , wherein said bonding reactions between each of said labeled detector molecules and said biopolymer are carried out simultaneously or successively. 
     
     
       33. The method of  claim 26 , wherein said bonding reaction in step (b) is selected from the group consisting of nucleic acid hybridization and an antigen/antibody reaction. 
     
     
       34. The method of  claim 33 , wherein said nucleic acid hybridization is an in situ hybridization. 
     
     
       35. The method of  claim 26 , wherein said biopolymers are selected from the group consisting of nucleic acids and polypeptides. 
     
     
       36. The method of  claim 35 , wherein said nucleic acids are DNA or RNA. 
     
     
       37. The method of  claim 26 , wherein said nucleic acids are chromosomal DNA. 
     
     
       38. The method of  claim 26 , wherein the labeled detector molecules are nucleic acids or antibodies. 
     
     
       39. The method of  claim 38 , wherein said different nucleic acids are selected from different chromosome region-specific DNA libraries. 
     
     
       40. The method of  claim 26 , wherein the label comprises a fluorescent dye. 
     
     
       41. The method of  claim 26 , wherein said step (a) further comprises providing at least one set of a localized calibrating probe, said probe comprising calibrating labels. 
     
     
       42. The method of  claim 41 , wherein said calibrating labels comprise all of said labels of said labeled detector molecules of the different sets. 
     
     
       43. The method of  claim 26 , wherein said step (a) further comprises providing a number of localized calibrating probes, said number being one less than the total number of said labels in said labeled detector molecules, each of said probes comprising two labels; and said step (d) further comprises correcting registration errors between individual images corresponding to individual labels, said registration errors being introduced by changing filters between the acquisition of said individual images; said correcting step being achieved by pairwise comparison of the positions of the two labels of said calibrating probes. 
     
     
       44. The method of  claim 43 , wherein said step (d) further comprises forming images of said biopolymers; and aligning said images with respect to said bondings, thereby providing positional correction for said bondings. 
     
     
       45. The method of  claim 44 , wherein said calibrating probes are fluorescence-labeled particles. 
     
     
       46. The method of  claim 44 , wherein said calibrating probes are used for positional correction of said bondings. 
     
     
       47. The method of  claim 43 , wherein said step (a) further comprises providing a plurality of said calibrating probes; and said step (d) further comprises correcting positional transformations of said bondings by comparison of the position of the labels of said calibrating probes. 
     
     
       48. The method of  claim 47 , wherein said step of correcting is automatic. 
     
     
       49. The method of  claim 47 , wherein said labels of said calibrating probes have known or reproducible constant intensity whereby the signal intensities of all of said labels can be standardized. 
     
     
       50. The method of  claim 49 , wherein said calibrating probes are fluorescence-labeled probes.